Artificial intelligence assisted compositional analyses of human abdominal aortic aneurysms ex vivo.

Quantification of histological information from excised human abdominal aortic aneurysm (AAA) specimens may provide essential information on the degree of infiltration of inflammatory cells in different regions of the AAA. Such information will support mechanistic insight in AAA pathology and can be linked to clinical measures for further development of AAA treatment regimens. We hypothesize that artificial intelligence can support high throughput analyses of histological sections of excised human AAA. We present an analysis framework based on supervised machine learning. We used TensorFlow and QuPath to determine the overall architecture of the AAA: thrombus, arterial wall, and adventitial loose connective tissue. Within the wall and adventitial zones, the content of collagen, elastin, and specific inflammatory cells was quantified. A deep neural network (DNN) was trained on manually annotated, Weigert stained, tissue sections (14 patients) and validated on images from two other patients. Finally, we applied the method on 95 new patient samples. The DNN was able to segment the sections according to the overall wall architecture with Jaccard coefficients after 65 epocs of 92% for the training and 88% for the validation data set, respectively. Precision and recall both reached 92%. The zone areas were highly variable between patients, as were the outputs on total cell count and elastin/collagen fiber content. The number of specific cells or stained area per zone was deterministically determined. However, combining the masks based on the Weigert stainings, with images of immunostained serial sections requires addition of landmark recognition to the analysis path. The combination of digital pathology, the DNN we developed, and landmark registration will provide a strong tool for future analyses of the histology of excised human AAA. In combination with biomechanical testing and microstructurally motivated mathematical models of AAA remodeling, the method has the potential to be a strong tool to provide mechanistic insight in the disease. In combination with each patients' demographic and clinical profile, the method can be an interesting tool to in supportof a better treatment regime for the patients.

Assessing Collagen and Elastin Pressure-dependent Microarchitectures in Live, Human Resistance Arteries by Label-free Fluorescence Microscopy.

The pathogenic contribution of resistance artery remodeling is documented in essential hypertension, diabetes and the metabolic syndrome. Investigations and development of microstructurally motivated mathematical models for understanding the mechanical properties of human resistance arteries in health and disease have the potential to aid understanding how disease and medical treatments affect the human microcirculation. To develop these mathematical models, it is essential to decipher the relationship between the mechanical and microarchitectural properties of the microvascular wall. In this work, we describe an ex vivo method for passive mechanical testing and simultaneous label-free three-dimensional imaging of the microarchitecture of elastin and collagen in the arterial wall of isolated human resistance arteries. The imaging protocol can be applied to resistance arteries of any species of interest. Image analyses are described for quantifying i) pressure-induced changes in internal elastic lamina branching angles and adventitial collagen straightness using Fiji and ii) collagen and elastin volume densities determined using Ilastik software. Preferably all mechanical and imaging measurements are performed on live, perfused arteries, however, an alternative approach using standard video-microscopy pressure myography in combination with post-fixation imaging of re-pressurized vessels is discussed. This alternative method provides users with different options for analysis approaches. The inclusion of the mechanical and imaging data in mathematical models of the arterial wall mechanics is discussed, and future development and additions to the protocol are proposed.

Imaging and modeling of acute pressure-induced changes of collagen and elastin microarchitectures in pig and human resistance arteries.

The impact of disease-related changes in the extracellular matrix (ECM) on the mechanical properties of human resistance arteries largely remains to be established. Resistance arteries from both pig and human parietal pericardium (PRA) display a different ECM microarchitecture compared with frequently used rodent mesenteric arteries. We hypothesized that the biaxial mechanics of PRA mirror pressure-induced changes in the ECM microarchitecture. This was tested using isolated pig PRA as a model system, integrating vital imaging, pressure myography, and mathematical modeling. Collagenase and elastase digestions were applied to evaluate the load-bearing roles of collagen and elastin, respectively. The incremental elastic modulus linearly related to the straightness of adventitial collagen fibers circumferentially and longitudinally (both R2 ≥ 0.99), whereas there was a nonlinear relationship to the internal elastic lamina elastin fiber branching angles. Mathematical modeling suggested a collagen recruitment strain (means ± SE) of 1.1 ± 0.2 circumferentially and 0.20 ± 0.01 longitudinally, corresponding to a pressure of ~40 mmHg, a finding supported by the vital imaging. The integrated method was tested on human PRA to confirm its validity. These showed limited circumferential distensibility and elongation and a collagen recruitment strain of 0.8 ± 0.1 circumferentially and 0.06 ± 0.02 longitudinally, reached at a distending pressure below 20 mmHg. This was confirmed by vital imaging showing negligible microarchitectural changes of elastin and collagen upon pressurization. In conclusion, we show here, for the first time in resistance arteries, a quantitative relationship between pressure-induced changes in the extracellular matrix and the arterial wall mechanics. The strength of the integrated methods invites for future detailed studies of microvascular pathologies.NEW & NOTEWORTHY This is the first study to quantitatively relate pressure-induced microstructural changes in resistance arteries to the mechanics of their wall. Principal findings using a pig model system were confirmed in human arteries. The combined methods provide a strong tool for future hypothesis-driven studies of microvascular pathologies.

Biochemical and Bioimaging Evidence of Cholesterol in Acquired Cholesteatoma.

OBJECTIVES: To quantify the barrier sterols and image the lipid structures in the matrix of acquired cholesteatoma and compare the distribution with that found in stratum corneum from normal skin, with the goal to resolve their potential influence on cholesteatoma growth. METHODS: High-performance thin-layer chromatography (HPTLC) was used to achieve a quantitative biochemical determination of the sterols. The intercellular lipids were visualized by Coherent Anti-Stokes Raman scattering (CARS) microscopy, which enables label-free imaging of the lipids in intact tissue samples. RESULTS: The results show that the total lipid content of the cholesteatoma matrix is similar to that of stratum corneum from skin and that the cholesteatoma matrix unquestionably contains cholesterol. The cholesterol content in the cholesteatoma matrix is increased by over 30% (w/w dry weight) compared to the control. The cholesterol sulfate content is below 1% of the total lipids in both the cholesteatoma and the control. Cholesterol ester was reduced by over 30% when compared to the control. CONCLUSIONS: The content of cholesterol in the cholesteatoma matrix is significantly different from that in stratum corneum from skin, and we confirm that the main structure of the cholesteatoma resembles very thick stratum corneum.

Quadrupolar, emission-tunable π-expanded 1,4-dihydropyrrolo[3,2-b]pyrroles--synthesis and optical properties.

The synthesis and optical characterization of six novel heteroaromatic-based chromophores is described. The new dyes present mostly an A-D-A general framework, where A is an electron-deficient aromatic ring and D is an electron-rich pyrrolo[3,2-b]pyrrole moiety, linked via triple bonds. It was demonstrated that the increase in the molecular length of the chromophore effectively extends π-conjugation. The effect of structural variations on photophysical properties was studied in detail for these compounds and the relationship between the structure and photophysical properties was thoroughly elucidated by comparison with simpler tetraaryl-analogues. The strong charge-transfer characteristic of these functional dyes can be illustrated by large Stokes shifts (4100-7100 cm(-1)) for A-D-A architectures. The replacement of phenyl rings at positions 2 and 5 with the arylethynylaryl substituents bathochromically shifts both absorption and emission at ca. 50-150 nm. The clear dependence of fluorescence maxima on the electron-accepting property of the peripheral arylethynyl substituent emphasizes strong π-conjugation in these molecules. The donor-acceptor interactions were also found to influence the two-photon absorption properties.

Tetraaryl-, pentaaryl-, and hexaaryl-1,4-dihydropyrrolo[3,2-b]pyrroles: synthesis and optical properties.

Efficient conditions for the synthesis of tetra-, penta-, and hexasubstituted derivatives of 1,4-dihydropyrrolo[3,2-b]pyrrole were developed. The tetraaryl derivatives were obtained in a novel one-pot reaction among aromatic aldehydes, aromatic amines, and butane-2,3-dione. After a thorough examination of various reaction parameters (solvent, acid, temperature) p-toluenesulfonic acid was identified as the crucial catalyst. As a result, 1,4-dihydropyrrolo[3,2-b]pyrroles were obtained in the highest yields reported to date. The scope and limitation studies showed that this new method was particularly efficient for sterically hindered aldehydes (yields 45-49%). Pentaaryl- and hexaaryl-1,4-dihydropyrrolo[3,2-b]pyrroles were prepared from tetraaryl-1,4-dihydropyrrolo[3,2-b]pyrroles via direct arylation by employing both electron-poor and electron-rich aromatic and heteroaromatic haloarenes. Strategic placement of electron-withdrawing substituents at the 2-, 3-, 5-, and 6-positions produced an acceptor-donor-acceptor type fluorophore. The resulting multiply substituted heteropentalenes displayed intriguing optical properties. The relationship between the structure and photophysical properties for all compounds were directly compared and thoroughly elucidated. All synthesized products displayed strong blue fluorescence and exhibited moderate to large Stokes shifts (3000-7300 cm(-1)) as well as high quantum yields of fluorescence up to 88%. Two-photon absorption cross-section values measured in the near-IR region were surprisingly high (hundreds of GM), given the limited conjugation in these propeller-shaped dyes.